REPLICATION INITIATES AT A CONFINED REGION DURING DNA AMPLIFICATION IN SCIARA DNA PUFF II-9A

Two independent two-dimensional (2D) gel methods were used to map an o rigin of replication that is developmentally regulated by the steroid hormone ecdysone, namely an origin for DNA puff amplification in the f ungus fly Sciara coprophila. Initiation of replication was found to oc cur within a small region of no larger than 6 kb by use of the neutral /neutral 2D gel method. Neutral/alkaline 2D get analyses support the r esults of the neutral/neutral 2D gels and further define within the or igin region an approximately 1-kb area where the majority of replicati on initiates. This is the first example of an origin of replication in multicellular eukaryotes that has been mapped by 2D gels to such a sm all defined region. Moreover, replication can be seen by the neutral/a lkaline 2D gel method to proceed bidirectionally outward from this rep lication origin region. These data are consistent with an onion-skin m echanism whereby multiple rounds of DNA replication initiate at a spec ific origin of replication for Sciara DNA puff amplification.